In vivo fluorescence imaging has seen considerable progress over the last several years. During the previous 3 years of funding (2006-2009) we have introduced a number of advances to whole animal fluorescence tomographic (FMT) imaging: 1) we have developed theoretical and technology frameworks to perform non- fluid, non-fiber system FMT (free-space imaging), 2) we have designed algorithm to image with high fidelity in optically heterogeneous, diffuse media, 3) we have implemented fast inversion methods that allow reconstructions of large data sets in seconds-minutes, 4) we have developed hybrid imaging approaches incorporating computed tomography (CT) or magnetic resonance imaging (MRI), 5) we have constructed a prototype hybrid CT-FMT imaging system and 6) we have developed multichannel FMT. There are a number of unique opportunities to further advance FMT imaging and apply it to important biomedical questions. In particular, the recent discovery of two new reporter probe platforms (upconverting nanoparticles (UNP) and far red fluorescent proteins, RFP) are likely to have considerable impact. Upconverting nanoparticles promise to essentially eliminate background autofluorescence by shifting illumination to 980 nm. RFP on the other hand, will allow the quantization of any protein of interest (potentially even pathways, cellular processes and protein- protein interactions). In order to harness the full power of these newer reporters, new reconstruction algorithm, experimental set-ups and rigorous validation against accepted gold-standards are required. The goal of this proposal is therefore to adapt current FMT instrumentation and algorithm to the new reporters, rigorously validate them in phantoms and apply them to highly relevant mouse models of disease. In the first aim (RFP imaging), we will address three sub-topics: a) a systematic and rigorous comparison of different RFP tumor cell lines for in vivo imaging, b) iteratively adapting the reconstruction algorithm for RFP based on the above tissue measurements and c) using the optimized approach to quantitate cell mass in vivo. In a second aim (UNP imaging) we will adapt and optimize FMT-CT reconstruction algorithm for the detection of UNP in vivo and developing targeted combined PET-FMT agents based on the UNP platform. In this resubmission we have a) obtained extensive new data on the feasibility of PET-FMT fusion experiments, b) have published another 8 manuscripts on hybrid FMT and/or UNP imaging and c) have further clarified minor concerns raised during the previous review. We believe that these new approaches will offer vastly higher sensitivity and accuracy, simplify experimental procedures and allow a seamless integration with other imaging modalities and biological data sets.